50 results about "DNA Integration" patented technology

A process for integrating a DNA fragment into the genome of a cell of a monocotyledonous plant, the process comprising the steps of: 1) incubating, prior to contacting with the DNA fragment, a culture of untransformed monocotyledonous plant cells on a medium comprising a plant phenolic compound, for a period of time sufficient to stimulate cell division and enhance competence for integration of foreign DNA; and 2) contacting the untransformed cells with the DNA fragment under conditions in which the DNA fragment is taken up by the untransformed cells and is stably integrated in the genome of the untransformed cells, to generate transformed cells.

Described are recombinant yeast which ferment xylose to ethanol and which maintain their ability to do so when cultured for numerous generations in non-selective media. The preferred yeast contain multiple copies of integrated genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase fused to promoters which are non-glucose inhibited and which do not require xylose for induction. Also described are preferred methods for integrating multiple copies of exogenous DNA into host cells by transforming cells with replicative/integrative vectors, and then replicating the cells a number of times under selective pressure to promote retention of the vector in subsequent generations. The replicated vectors thus serve to integrate multiple copies of the exogenous DNA into the host cells throughout the replication/selection phase. Thereafter the selective pressure can be removed to promote loss of the vector in subsequent generations, leaving stable integrants of the exogenous DNA.

The invention relates to a method for cultivating and producing polygalacic acid with polygala root hair-shaped roots, which uses polygala root hypocotyl and blades as an explantation and converts by activating hair-shaped root agrobacterium, and leads T-DNA of hair-shaped root agrobacterium Ri plasmid to be integrated to polygala root nucleus DNA for expressing and forming hair-shaped roots, and polygala root hair-shaped roots are gained through plural times of subinoculation. The polygala root hair-shaped roots which are gained contain the same components of polygalacic acid (namely, senega-saponin) as that of traditional Chinese medicine polygala root, and the content is not lower than the wild traditional Chinese medicine polygala root. The invention can be used as new medicine resources to provide polygala root products, which solves the practical problems that the demand amount of polygala root is increased year by year, and wild resources are lacked and the quality of cultivated breeds is reduced, which brings practical problems to clinic, Chinese patent drug processes and export.

A novel mini-retrotransposon vector system is provided for integrating foreign DNA into plants. The invention includes a novel vector comprising a truncated and modified retroelement which includes the 5′ and 3′ LTR regions that provide transcription initiation and termination sites as well as the cis acting sequences required for reverse transcription. Novel vectors, plant cells, and methods of using the same are disclosed.

The invention relates to a method for in-situ transgenosis of corn mature embryos. The method comprises the following steps: obtaining germs of mature embryos needed for in-situ transgenosis of the corn mature embryos; treating the germs to expose growing points and slightly damaging the growing points; carrying out in-situ transgenosis in the growing points of the corn by utilizing impregnating fluid; coculturing impregnated cells at the growth points and agrobactrium tumefaciens to enable Bar genes to be integrated into corn chromosomes by virtue of T-DNA (triplex-deoxyribonucleic acid) integration of the agrobactrium tumefaciens and rejuvenating the Bar genes; carrying out herbicide screening to initially obtain a herbicide resistant plant for in-situ transgenosis and transplanting the plant; obtaining genetically modified corn seeds by selfing pollination of a PCR (polymerase chain reaction) positive plant of the in-situ genetically modified corn. According to the method provided by the invention, transgenosis is carried out on the growth points of the corn mature embryos and the method of spraying or coating herbicide on corn lamina to screen transgenic plants and carrying out molecular detection to further verify the PCR positive plants is adopted. The method provided by the invention is simple and efficient and can secure a conversion rate of 4%, thus being of great significance for genetic engineering and inheritance breeding of corn.

The invention discloses a screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells. The screening report vector includes a universal sgRNA expression cassette, a Cas9 expression cassette, a resistance gene expression cassette and a resistance gene homologous recombination repair template, and the screening reporter vector itself can be repaired by homologous recombination of the resistance gene sequence to make the cells have resistance. The screening report vector, a targeting vector for cell genome HDR editing and a donor vectorform a co-transfection system which can be applied to HDR positive cell cloning with efficient enrichment point editing, fragment insertion and fragment deletion, and can be widely applied to a variety of cells of mammals. The screening efficiency of HDR-edited cells is significantly improved, the reintroduction of double-strand breaking and DNA integration in the unrelated positions of the cellgenome is not required, and an effective way is provided to promote research and application of accurate gene editing.

The invention discloses recombinant pseudomonas putida, which is expressed on the basis of DNA integration. The invention also discloses a construction method for the recombinant pseudomonas putida, and the application of the recombinant pseudomonas putida in phosphorus removal from wastewater. The method of the invention is simple, and obtained genetically engineered bacteria have high-efficiency phosphorus removal capacity.

A novel protein having inhibitory activity on the protease activity of hepatocyte growth factor (HGF) activator was purified and isolated, and its molecular weight (ca. 40,000 daltons) and its N-terminal and partial amino acid sequences were determined. A gene coding for the protein was cloned, and the gene DNA was incorporated into a vector, for transforming host cells. Cultivation of the transformant gave the desired protein. The protein can be used as an in vivo or in vitro control factor for HGF or HGF activator. It is also useful as an antigen to be used in producing an antibody to be used as means for kinetic studies of the protein, or as a standard in assay systems therefor.

A DNA encoding a novel enzyme which has an activity of synthesizing D-serine from glycine and formaldehyde; a recombinant DNA constructed by integrating the DNA into a vector; a transformant having been transformed by the recombinant DNA; and a method of producing D-serine from glycine and formaldehyde by using the above enzyme.

A method for detecting, in liver tissue isolated from a human subject and suspected of being cancerous, integration of HBV-DNA into the MLL4 gene, this integration of HBV-DNA indicating the cancerous nature of a tissue. In a typical case, this integration can be detected by carrying out PCR using a primer specific to the region containing intron 3 of the MLL4 gene and a primer specific to the X gene region of HBV.

The invention is for a DNA vaccine expressing the hemagglutinin (HA1) gene of equine-2 influenza virus. By engineering a stop codon within HA1, expression of HA1 is ensured. By encapsulation of the DNA vaccine in liposome and by intranasal inoculation, it is sufficient to elicit protective immunity at a significantly lower dosage compared to a DNA vaccine expressing the full length HA gene. Lower dosage reduces the risk of induction of anti-DNA antibodies. Intranasal inoculation directly to the respiratory epithelial cells reduces the risk of DNA integration. The inventive vaccine is advantageous over current inactivated or live attenuated vaccines, as updating of the vaccine requires only the replacement of the encoding sequence with the new virus.

The invention discloses a method for breeding cattle capable of producing hypoallergenic milk and application of the method. The method comprises the following steps: leading a zinc finger nuclease carrier pZFN into fibroblasts of the cattle, so as to obtain donor cells with a gene type which is a biallelic mutation type, wherein the biallelic mutation type is that nucleotide molecules of beta-LGgenes on two homologous chromosomes of the cattle are subjected to frame shift mutation; transferring cell nucleuses of the donor cells into cattle oocytes without the cell nucleuses and developing toform reconstructed embryos; then transferring the reconstructed embryos into uteri of cows and delivering to obtain the cattle capable of producing the hypoallergenic milk. According to the method disclosed by the invention, beta-LG biallelic gene knockout cattle which have no exogenous DNA (Deoxyribonucleic Acid) integration, can produce beta-lactoglobulin-free milk and deliveries mutation to the next generation through normal breeding are successfully bred. By adopting the method disclosed by the invention, an important foundation is laid for allergenic problems of the milk and also provides infinite possibility for preparing humanized milk. The method has important application value.

The invention relates to construction of an 18-trisomy syndrome cell model and a cell bank of 18-trisomy syndrome cell by SV40LT gene transfection for the field of medical research. The construction is mainly characterized in that an SV40LTag-pcDNA3.1(-) recombinant is constructed through a T4DNA ligase, BamHI, pcDNA3.1(-) DNA and SV40LTag DNA as a matter of routine, the recombinant is purified through competent escherichia coli, the recombinant is introduced into an in-vitro passage cell digested through collagenase II or an 18-trisomy syndrome tissue cell which is logarithmically grown through a lipoid transfection method to be integrated with cell DNA, and a cloned cell which is screened through G418 and contains a positive recombinant is subjected to amplification culture; and a cell which accords with the characteristics of an immortalized cell and is the same as or similar to a primary cell on a cell morphology, a growth curve, a chromosome karyotype, soft agar colony growth, a nude mouse tumorigenesis test, SV40 large T gene detection of transfection cell DNA, an mRNA expression product measurement result and a DNA sequence measurement result is screened as the 18-trisomy syndrome in-vitro research cell model and cryopreserved in liquid nitrogen, so that the foundation is laid for researching the pathogenesis of 18-trisomy syndrome for a long time from cell level in vitro.

Cellular targets for anti-retroviral drug development are disclosed. The cellular targets comprise ATR kinase and its relevant substrates, based on the identification of the ATR kinase as required for the final step of retroviral DNA integration. Assays for identifying modulators of retroviral integration via the ATR kinase pathway are disclosed, as well as modulators identified by such assays. Pharmaceutical preparations and methods of their use in treating retroviral infection are also disclosed.

The invention relates to a method for constructing hTERT mediated T lymphocytic model and a cell bank thereof in the field of medicines. The method is mainly characterized by comprising the following steps: carrying out double enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo with incision enzymes EcoR I and Xho I, connecting hTERT and pLXSNneo digested products subjected to PCR amplification and gel electrophoresis separation by virtue of Ligation Mix to construct a pLXSNneo-hTERT recombinant, transforming DH5a competent cells for purifying, amplifying and selecting ampicillin-resistant bacterial colony extraction plasmids, and performing in-vitro subculture on T lymphocytes in logarithmic growth by virtue of lipofection transfection, so that the recombinant is integrated with DNA of cells; performing enlarged culture, performing G418 screening on cells containing positive recombinants, screening cells of which the cellular morphology, growth curve, karyotype, inoculated nude mice test, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycle and apoptosis rate accord with the characteristics of immortalized cells and which are the same or similar to primary cells to serve as an hTERT mediated in-vitro T lymphocytic research model so as to be cryopreserved in liquid nitrogen. Therefore, a foundation is laid for long-term in-vitro research of pathogenesis of related diseases from the cellular level.

A method of modifying a target region in a host DNA using a donor DNA:

• • wherein the donor DNA having regions homologous to a 5′-side region outside of the target region in the host DNA, a 3′-side region outside of the target region in the host DNA and a first homologous recombination region inside of the target region in the host DNA, respectively, in this order, and further having a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region;
• which method has the steps of:
  • a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5′-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and
  • a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, to conduct selection of a host whose target region is modified based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter: and a selectable marker cassette for use in the method.

A method for producing a protein of interest on a manufacturing scale is based on integration, by homologous recombination, of the DNA encoding the protein of interest into a bacterial cell genome at a pre-selected site. The manufacturing scale production of recombinant proteins is in the fed-batch mode, semi-continuous or in a chemostat.

The invention relates to building of an SV40LT antigen gene-mediated klinefelter syndrome cell model and a cell bank of the klinefelter syndrome cell model, applied to the field of medical research. The building method is mainly characterized by comprising the following processes: building an SV40LTag-pcDNA3.1(1) recombinant by using T4DNA ligase, BamHI, pcDNA3.1(1) DNA and SV40LTag DNA according to a conventional method; purifying the recombinant by using competent escherichia coli, introducing the recombinant into an in vitro passage cell which is digested by collagenase II, or klinefelter syndrome histocyte which is in logarithmic growth, and integrating the recombinant with DNA; carrying out enlarging cultivation, screening cells containing positive recombinants by G418, and cloning; and screening a cell of which the cell morphology, the growth curve, karyotype, soft agar colony growth, nude mice tumor experiment result and transfection cell SV40 big T gene detection result, mRNA product measuring result and DNA sequencing result are in fit with the characteristics of an immortalized cell and are the same as or similar to that of a primary cell to be taken as an in vitro research cell model for the SV40LT-mediated klinefelter syndrome; and a foundation is laid for in vitro long-term research of pathogenesis of the klinefelter syndrome from the cellular level.

The invention relates to construction of an SV40LT gene-mediated megakaryocyte model and a cell bank of megakaryocyte, belonging to the field of medical research. The construction is mainly characterized in that an SV40LTag-pcDNA3.1 (-) recombinant is constructed from T4DNA ligase, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA by virtue of conventional means; the recombinant is purified by virtue of competent escherichia coli; the recombinant is introduced into megakaryocyte cultured in vitro through a lipofection transfection method, so that the recombinant is integrated with the DNA of the cell; a cell screened by virtue of G418 and containing a positive recombinant is subjected to subculture and enlarged cultivation; a cell which is consistent with the biological properties of an immortalized cell in cellular morphology, cell growth curve, karyotype, nude mice carcinogenicity test, detection of SV40 large T gene in transfection cell DNA, determination of an mRNA expression product and a DNA sequence determination result is screened out as a megakaryocyte model, and is cryopreserved in liquid nitrogen, so as to construct an SV40LT gene-mediated megakaryocyte model and the cell bank of megakaryocyte; therefore, a method for long-term in vitro subculture of the megakaryocyte is established, and the method is applicable to the in vitro research of development, function and pathogenic mechanism of the megakaryocyte.

The present invention provides a system for site-specific directed gene insertion of desired genes or foreign DNA into cellular genomes. The system includes novel vectors for integrating DNA into the genome of different hosts. Methods of using the vectors and transformed hosts are described.